Selective and reactive protease inhibitors are widely used in biomedical research. They are used to identify the nature of new proteolytic enzymes encountered in isolation and characterization studies. They are also used both in research and industrially to prevent undesired proteolysis of proteins and peptides during the production, isolation, purification, transport and storage of these valuable materials. They could also be used industrially to increase the yields of proteins and peptides produced by cloning or fermentation by inhibiting proteolysis of the products due to proteases released by the parent organism or by contaminating organisms in the growth medium. Protease inhibitors could also be used to protect proteins and peptides during storage or use from proteolysis which would destroy or alter the activity and/or function of the peptides or proteins. Such uses would include their addition to antibodies, enzymes, plasma proteins or other proteins which are widely sold for use in clinical analysis, biomedical research, therapy, and for many other reasons.
Serine proteases are the most widely distributed class of proteolytic enzymes. Serine proteases can be further divided into three main classes based on their substrate specificity: chymotrypsin-like, elastase-like, and trypsin-like. In addition, there are a few other serine proteases which do not fit into any of the above three groups. This invention relates only to chymotrypsin-like and elastase-like enzymes. Chymotrypsin and chymotrypsin-like enzymes normally cleave peptide bonds in proteins and peptides where the amino acid residue on the carbonyl side of the split bond (P.sub.1 residue) is typically Trp, Tyr, Phe, Met, Leu or other amino acid residues which contain aromatic or large alkyl side chains. Elastase and elastase-like enzymes, on the other hand, cleave peptide bonds where the P.sub.1 amino acid residue is much smaller, typically Ala, Val, Ser, Leu and other similar amino acids. All of the above enzymes have extensive secondary specificity and recognize amino acid residues removed from the P.sub.1 residue. Chymotrypsin-like and elastase-like enzymes are widely distributed. They are found in leukocytes (white blood cells), mast cells, pancreatic juice, and many other cell types and fluids in higher organisms. They are also secreted by many types of bacteria, yeast, viruses and parasites. These enzymes can be very destructive if not properly controlled. Thus there is a need for selective and reactive inhibitors for these enzymes.
Sulfonyl fluorides have been widely used as inhibitors of serine proteases since their intial discovery by Fahrney and Gold (cf. J. Am. Chem. Soc. 85, pp. 997-1000 (1963)). These reagents, which are fairly stable in aqueous solution, inhibit serine proteases by sulfonylation of the active site serine residue to give a stable sulfonyl enzyme derivative. Sulfonyl fluorides have been shown to inhibit most serine proteases including chymotrypsin (cf. Baker and Hurlbut, J. Med. Chem., 12, pp 118-122 (1969)), trypsin, elastase, and complement, coagulation, and fibrinolytic serine proteases. However, sulfonyl fluorides are not usually considered to be highly specific and in fact phenylmethanesulfonyl fluoride is usually considered to be a fairly general inhibitor of serine proteases. Lively and Powers showed that the specificity of sulfonyl fluorides toward elastase, cathepsin G and chymotrypsin could be improved with structural changes in the inhibitor (cf. Biochim. Biophys. Acta 525, pp 171-179(1978)). While a difference in rate of 26,000 fold was observed between the fastest and slowest inhibitor, the absolute reactivities of the inhibitors were comparatively slow. It is an object of this invention to find a novel group of aryl sulfonyl fluorides which will rapidly and selectively inhibit elastase-like and chymotrypsin-like enzymes.